Piggybac transposon variants and methods of use

a technology of piggybac and variants, applied in the field of piggybac transposon variants and methods of use, can solve the problems of method limitations, size constraints of dna condensing reagents and virus-mediated strategies, and the limited amount of nucleic acids that can be transfected into cells, so as to achieve a lower rate of integration

Inactive Publication Date: 2011-12-22
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
View PDF0 Cites 27 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]In one embodiment, the integration defective piggyBac transposon ...

Problems solved by technology

However, such methods can have certain limitations.
For example, there are size constraints associated with DNA condensing reagents and virus-mediated strategies.
Further, the amount of nucleic acid that can be transfected into a cell is limited in virus strategies.
Not all methods facilitate insertion of the delivered nucleic acid into cellular nucleic acid, and while DNA condensing methods and lipid-containing reagents are relatively easy to prepare, the ...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Piggybac transposon variants and methods of use
  • Piggybac transposon variants and methods of use
  • Piggybac transposon variants and methods of use

Examples

Experimental program
Comparison scheme
Effect test

example 1

Identification of Integration-Defective PiggyBac Variants

[0191]The present experiments describe screening and identification of excision-hyperactive piggyBac transposons using a version of the Cherry gene which produces a red fluorescent protein. A copy of the piggyBac transposon was put into the gene, inactivating it such as the cells are NOT red. However, piggyBac excision restores the gene, leading to the production of the red fluorescent protein. Accordingly, increased red colony color identifies mutants that excise better.

[0192]A large collection of mutant transposase genes was made using mutagenic PCR which was cloned into an expression vector in yeast. Colonies containing individual mutants were grow up on an agar plate and were then examined with red fluorescent light. FIG. 4 shows excision hyperactives that have been isolated to date.

[0193]In certain preferred embodiments, the integration defective piggyBac comprises an amino acid change in the wild type piggyBac sequence c...

example 2

Identification of Hyperactive Variants

[0195]Using the integration defective piggyBac mutants as a starting point, the present inventors have identified hyperactive piggyBac transposon mutants. The yeast excision assay that was developed as described in Mitra R. et al. (piggyBac can bypass DNA synthesis during cut and paste transposition. EMBO J. Apr 9; 27(7):1097-109. Epub 2008 Mar. 20) was used to identify the hyperactive mutants. The piggyBac ORF was mutagenized by mutagenic PCR using primers flanking the ORF as the expression construct and then recovered transformants by co-transformation of the PCR product with a gapped piggyBac plasmid into the yeast assay strain containing a ura− to ura+ cassesette in which transposon excision results in formation of ura+ colonies. Following recovery of transformants on SC-Trp-His plates, colonies were resuspended in water and spotted onto plates lacking uracil to identify excisions. By comparison to the number of ura+ colonies from the mutage...

example 3

Induced Pluripotent Stem Cell Generation Using the Hyperactive Transposon

[0198]In certain exemplary embodiments, the hyperactive piggyBac transposons can be used to created induced pluripotent stem cells using a minimal set of genes. In particular, Oct 3 / 4, Sox2, Klf4 and c-myc are used as a minimal set of genes. Takahashi et al. (Cell, 131, 861-872, Nov. 30, 2007), incorporated by reference in its entirety herein, teach methods of generating induced pluripotent stem cells (iPS) from human dermal fibroblasts using Oct 3 / 4, Sox2, klf4, and c-Myc.

Other Embodiments

[0199]From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0200]The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Nucleic acid sequenceaaaaaaaaaa
Levelaaaaaaaaaa
Login to view more

Abstract

The present invention provides hyperactive piggyBac transposons, in particular hyperactive piggyBac transposons from Trichoplusia ni (cabbage looper moth) that transpose at a higher frequency than wildtype. The invention also features integration defective piggyBac transposons. The piggyBac transposons and transposases can be used in gene transfer systems for stably introducing nucleic acids into the DNA of a cell. The gene transfer system can be used in methods, for example, but not limited to, gene therapy, insertional mutagenesis, or gene discovery.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 155,207, filed on Feb. 25, 2009, the entire contents of which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Typical methods for introducing DNA into a cell include DNA condensing reagents such as calcium phosphate, polyethylene glycol, lipid-containing reagents, such as liposomes, multi-lamellar vesicles, as well as virus-mediated strategies. However, such methods can have certain limitations. For example, there are size constraints associated with DNA condensing reagents and virus-mediated strategies. Further, the amount of nucleic acid that can be transfected into a cell is limited in virus strategies. Not all methods facilitate insertion of the delivered nucleic acid into cellular nucleic acid, and while DNA condensing methods and lipid-containing reagents are relatively easy to prepare, the insertion of nucleic acid into viral vectors can ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): A61K38/45C12N15/63C12N5/10C07H21/04C12N9/12
CPCA61K48/00C12N2800/00C12N15/90C12N15/85A61K38/45C12N9/1241C12Y207/07
Inventor CRAIG, NANCY LYNN
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap